Background

T regulatory Type 1 (Tr1) cells are peripheral CD4⁺FoxP3^– regulatory T cells expressing CD49b and LAG-3 in mice and humans. Mouse Tr1 cells inhibit antigen-presenting cell (APC) activation via secretion of IL-10 and TGF-β and via cell-contact dependent mechanisms mediated through their expression of programmed cell death (PD)-1. However, the phenotype and functions of Tr1 cells non-human primates are still unknown.

Method

Mononuclear cells were isolated from the peripheral blood of cynomolgus monkeys(PBMCs). First, co-expression of CD49b and LAG-3 was compared among bona fide CD4⁺CD25^highFoxP3⁺ Tregs and CD4⁺CD25^–Foxp3^–using flow cytometry. Second, cytokines(IL-10, TGF-β, IFN-γ, IL-4, IL-17 etc.,) were processed after polyclonal activation via anti-CD3/CD28 Ab-coated beads. Next, we compared PD-L1 and CD45RO expression by Tr1 cells collected from the PBMCs of naïve monkeys, monkeys undergoing rejection of kidney allografts and monkeys which have been rendered tolerant of kidney allografts via donor mixed hematopoietic chimerism induction and leukocyte costimulation blockade.

Results

In cynomolgus monkeys, CD49b and LAG-3 were co-expressed on CD4⁺CD25^–Foxp3^– Tr1lymphocytes but not on CD4⁺FoxP3⁺Tregs. After polyclonal activation via anti-CD3/CD28 Ab-coated beads, virtually all Tr1 cells secreted high levels of IL-10 anti-inflammatory cytokine but no pro-inflammatory IFN-γ or IL-17 cytokines. We observed a significant expansion of Tr1 cells expressing a memory phenotype (CD45RO) and the immunomodulatory receptor PD-L1 in tolerant but not other animals.

Conclusion

Our data show that the surface markers CD49b and LAG-3 can be used to distinguish Tr1 from “classical” FoxP3⁺ Tregs in cynomolgus monkeys. Tr1 cells are likely to inhibit T cell responses mediated by PD-1/PD-L1 interactions. The contribution of these activated Tr1 cells to induction and maintenance of organ allograft tolerance in non-human primates is under investigation.

CITATION INFORMATION: Yu R, Tonsho M, Spencer P, Bernard-Stoecklin S, Benichou G, Madsen J. Characterization of T Regulatory Type 1 (Tr1) Cells in Naïve and Transplanted Non-Human Primates. Am J Transplant. 2016;16 (suppl 3).

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